Mammalian cytokine-like factor 7

ABSTRACT

Novel mammalian zcyto7 polypeptides, polynucleotides encoding the polypeptides, and related compositions and methods including antibodies and anti-idiotypic antibodies.

This application is a continuation of U.S. application Ser. No.10/772,985, filed Feb. 5, 2004, which is a continuation of U.S.application Ser. No. 09/633,452, which is a continuation of U.S.application Ser. No. 09/066,745, filed Apr. 24, 1998, which claims thebenefit of U.S. Provisional Application Ser. No. 60/071,676 filed Jan.16, 1998; and U.S. Provisional Application Ser. No. 60/044,886 filedApr. 25, 1997.

BACKGROUND OF THE INVENTION

Proliferation and differentiation of cells of multicellular organismsare controlled by hormones and polypeptide growth factors. Thesediffusable molecules allow cells to communicate with each other and actin concert to form cells and organs, and to repair and regeneratedamaged tissue. Examples of hormones and growth factors include thesteroid hormones (e.g. estrogen, testosterone), parathyroid hormone,follicle stimulating hormone, the interleukins, platelet derived growthfactor (PDGF), epidermal growth factor (EGF), granulocyte-macrophagecolony stimulating factor (GM-CSF), erythropoietin (EPO) and calcitonin.

Hormones and growth factors influence cellular metabolism by binding toproteins. Proteins may be integral membrane proteins that are linked tosignaling pathways within the cell, such as second messenger systems.Other classes of proteins are soluble molecules, such as thetranscription factors.

Of particular interest are cytokines, molecules that promote theproliferation and/or differentiation of cells. Examples of cytokinesinclude erythropoietin (EPO), which stimulates the development of redblood cells; thrombopoietin (TPO), which stimulates development of cellsof the megakaryocyte lineage; and granulocyte-colony stimulating factor(G-CSF), which stimulates development of neutrophils. These cytokinesare useful in restoring normal blood cell levels in patients sufferingfrom anemia or receiving chemotherapy for cancer. The demonstrated invivo activities of these cytokines illustrates the enormous clinicalpotential of, and need for, other cytokines, cytokine agonists, andcytokine antagonists.

SUMMARY OF THE INVENTION

The present invention addresses this need by providing a novelpolypeptide called cytokine-like factor 7,hereinafter referred to asZcyto7 and related compositions and methods.

Thus, one aspect of the present invention provides for an isolatedZcyto7 polypeptide having amino acid sequences as follows. Both thehuman and the mouse cDNAs have been discovered. The human sequences aredefined by SEQ ID NOs: 1 and 2. The murine nucleotide and amino acidsequences are defined by SEQ ID NOs: 11 and 12.

The nucleotide sequence of SEQ ID NO:1 contains an open reading frameencoding a polypeptide of about 180 amino acids with the initial Met asshown in SEQ ID NO:1 and SEQ ID NO:2. A predicted signal sequence iscomprised of amino acid residues 1-20, and the resultant predictedmature Zcyto7 polypeptide is represented by the amino acid sequenceextending from amino acid residue 21, a glutamine to and including aminoacid residue 180 a phenylalanine, also represented by SEQ ID NO: 14.Peptide mapping data indicate that mature Zcyto7 can be comprised of anumber of N-terminal mature variants including the amino acid sequenceextending from amino acid residue 23, an arginine to and including aminoacid residue 180 of SEQ ID NO: 2, also defined by SEQ ID NO: 36; aminoacid sequence extending from amino acid residue 27, a serine to andincluding amino acid residue 180 of SEQ ID NO: 2, also defined by SEQ IDNO: 37; the amino acid sequence defined by amino acid residue 30, alysine, to and including amino acid residue 180 of SEQ ID NO: 2, alsodefined by SEQ ID NO: 38; amino acid sequence extending from amino acid28, a lysine, to and including amino acid residue 180 of SEQ ID NO:2,also defined by SEQ ID NO: 41 and the amino acid sequence extending fromamino acid residue 53, a methionine, to and including amino acid residue180, also defined by SEQ ID NO: 42. The only observed cleavage at thecarboxyl terminus is the phenylalanine at position 180 can be cleavedoff. This can occur in all of the above-defined mature Zcyto7polypeptides an example of which is shown by SEQ ID NO:43. Additionalvariants of human Zcyto7 are defined by SEQ ID NOs: 15-25. Within anadditional embodiment, the polypeptide further comprises an affinitytag.

SEQ ID NOs: 11 and 12 define murine Zcyto7 wherein the mature proteinextends from amino acid residues amino acid residue 21, a histidine, toand including amino acid residue 180 a phenylalanine, also defined bySEQ ID NO: 39; or as an alternative splice site from amino acid residue23, an arginine, to and including amino acid 180 also defined by SEQ IDNO: 40. The present invention is also comprised of polypeptides havingan amino acid sequence at least 90% identical, more preferably 95%, 97%or 99% identical to those Zcyto7 polypeptides defined in above.

An additional embodiment of the present invention relates to a peptideor polypeptide which has the amino acid sequence of an epitope-bearingportion of a Zcyto7 polypeptide having an amino acid sequence describedabove. Peptides or polypeptides having the amino acid sequence of anepitope-bearing portion of a Zcyto7 polypeptide of the present inventioninclude portions of such polypeptides with at least nine, preferably atleast 15 and more preferably at least 30 to 50 amino acids, althoughepitope-bearing polypeptides of any length up to and including theentire amino acid sequence of a polypeptide of the present inventiondescribed above are also included in the present invention. Examples ofsaid polypeptides are defined by the amino acid sequences of SEQ ID NOs:25-35. Also claimed are any of these polypeptides that are fused toanother polypeptide or carrier molecule.

The present invention further comprises a polypeptide defined by SEQ IDNOs: 15-25 wherein the amino termini of said polypeptides are modifiedand begin at either amino acid residue 3, an arginine; amino acidresidue 7, a serine; amino acid residue 8, a lysine; amino acid residue10, a lysine or amino acid residue 33 methionine.

The present invention is further comprised of a polypeptide wherein thepolypeptide is a polypeptide defined by SEQ ID NOs: 2, 12, 14-25 and 36to 42 wherein the amino acid sequences end at the isoleucines at aminoacid residue 179 of SEQ ID NO: 2, at amino acid residue 159 of SEQ IDNOs: 14-25, which corresponds to amino acid residue 157 of SEQ ID NO:36,amino acid residue 153 of SEQ ID NO:37, amino acid residue 150 of SEQ IDNO:38, amino acid residue 159 of SEQ ID NO: 39, amino acid residue 157of SEQ ID NO:40, amino acid residue 152 SEQ ID NO:42, amino acid residue127 of SEQ ID NO:42.

The present invention is further comprised of an isolated peptide orpolypeptide of the above-described peptides or polypeptide having anamino acid sequence modified by addition, deletion and/or replacement ofone or more amino acid residues and which maintains the biologicalactivity of said peptide or polypeptide.

Within a further aspect of the invention there is provided a chimericpolypeptide consisting essentially of a first portion and a secondportion joined by a peptide bond. The first portion of the chimericpolypeptide consists essentially of (a) a Zcyto7 polypeptide asdescribed above (b) allelic variants of the polypeptides describedabove. The second portion of the chimeric polypeptide consistsessentially of another polypeptide such as an affinity tag. Within oneembodiment the affinity tag is an immunoglobulin F_(C) polypeptide. Theinvention also provides expression vectors encoding the chimericpolypeptides and host cells transfected to produce the chimericpolypeptides.

Another aspect of the present invention provides for isolated nucleicacid molecules comprising a polynucleotide selected from the groupconsisting of: (a) a nucleotide sequence encoding the Zcyto7polypeptides described above; (b)a nucleotide sequence encoding thepolypeptides of SEQ ID NOs: 14-40 and (c) a nucleotide sequencecomplementary to any of to any of the nucleotide sequences in (a) or(b).

Further embodiments of the invention include isolated nucleic acidmolecules that comprise a polynucleotide having a nucleotide sequence atleast 90% identical, and more preferably 95%, 97%, 98%, or 99% identicalto any of the nucleotide sequences in (a), (b) or (c) above, or apolynucleotide which hybridizes under stringent hybridization conditionsto a polynucleotide having a nucleotide sequence of (a) (b) or (c)above. An additional nucleic acid embodiment of the present inventionrelates to an isolated nucleic acid molecule comprising an amino acid ofan epitope-bearing portion of a Zcyto7 polypeptide.

Within another aspect of the invention there is provided an expressionvector comprising (a) a transcription promoter; (b) a DNA segmentencoding a polypeptide described above, and (c) a transcriptionterminator, wherein the promoter, DNA segment, and terminator areoperably linked.

Within a third aspect of the invention there is provided a culturedeukaryotic cell into which has been introduced an expression vector asdisclosed above, wherein said cell expresses a protein polypeptideencoded by the DNA segment.

In another embodiment of the present invention is an isolated antibodythat binds specifically to a Zcyto7 polypeptide described above. Alsoclaimed is a method for producing antibodies which bind to a Zcyto7polypeptide comprising inoculating a mammal with a Zcyto7 polypeptide orZcyto7 epitope-bearing polypeptide so that the mammal producesantibodies to the polypeptide; and isolating said antibodies.

These and other aspects of the invention will become evident uponreference to the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION

The teachings of all of the references cited herein are incorporated intheir entirety herein by reference.

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A [Nilsson et al, EMBO J. 4:1075 (1985);Nilsson et al., Methods Enzymol. 198:3 (1991)], glutathione Stransferase [Smith and Johnson, Gene 67:31 (1988)], Glu-Glu affinity tag[Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4 (1985)],substance P, FLAG™ peptide (Hopp et al., Biotechnology 6:1204-10 (1988),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2: 95-107 (1991). DNAs encoding affinity tags are availablefrom commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

The term “allelic variant” denotes any of two or more alternative formsof a gene occupying the same chromosomal locus. Allelic variation arisesnaturally through mutation, and may result in phenotypic polymorphismwithin populations. Gene mutations can be silent (no change in theencoded polypeptide) or may encode polypeptides having altered aminoacid sequence. The term allelic variant is also used herein to denote aprotein encoded by an allelic variant of a gene.

The term “expression vector” denotes a DNA molecule, linear or circular,that comprises a segment encoding a polypeptide of interest operablylinked to additional segments that provide for its transcription. Suchadditional segments may include promoter and terminator sequences, andmay optionally include one or more origins of replication, one or moreselectable markers, an enhancer, a polyadenylation signal, and the like.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

The term “isolated”, when applied to a polynucleotide molecule, denotesthat the polynucleotide has been removed from its natural genetic milieuand is thus free of other extraneous or unwanted coding sequences, andis in a form suitable for use within genetically engineered proteinproduction systems. Such isolated molecules are those that are separatedfrom their natural environment and include cDNA and genomic clones.Isolated DNA molecules of the present invention are free of other geneswith which they are ordinarily associated, but may include naturallyoccurring 5′ and 3′ untranslated regions such as promoters andterminators. The identification of associated regions will be evident toone of ordinary skill in the art. See for example, Dynan and Tijan,Nature 316:774-78 (1985). When applied to a protein, the term “isolated”indicates that the protein is found in a condition other than its nativeenvironment, such as apart from blood and animal tissue. In a preferredform, the isolated protein is substantially free of other proteins,particularly other proteins of animal origin. It is preferred to providethe protein in a highly purified form, i.e., greater than 95% pure, morepreferably greater than 99% pure.

The term “operably linked”, when referring to DNA segments, denotes thatthe segments are arranged so that they function in concert for theirintended purposes, e.g. transcription initiates in the promoter andproceeds through the coding segment to the terminator

The term “polynucleotide” denotes a single- or double-stranded polymerof deoxyribonucleotide or ribonucleotide bases read from the 5′ to the3′ end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules.

The term “complements of polynucleotide molecules” denotespolynucleotide molecules having a complementary base sequence andreverse orientation as compared to a reference sequence. For example,the sequence 5′ ATGCACGGG 3′ is complementary to 5° CCCGTGCAT 3′.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

The term “promoter” denotes a portion of a gene containing DNA sequencesthat provide for the binding of RNA polymerase and initiation oftranscription. Promoter sequences are commonly, but not always, found inthe 5′ non-coding regions of genes.

The term “secretory signal sequence” denotes a DNA sequence that encodesa polypeptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger peptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule (i.e., a ligand) and mediates the effect of theligand on the cell. Membrane-bound receptors are characterized by amulti-domain structure comprising an extracellular ligand-binding domainand an intracellular effector domain that is typically involved insignal transduction. Binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell. This interactionin turn leads to an alteration in the metabolism of the cell. Metabolicevents that are linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids. Most nuclear receptors also exhibit amulti-domain structure, including an amino-terminal, transactivatingdomain, a DNA binding domain and a ligand binding domain. In general,receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g.,thyroid stimulating hormone receptor, beta-adrenergic receptor) ormultimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor,GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6receptor).

The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity of<10⁹ M⁻¹.

A “soluble protein” is a protein polypeptide that is not bound to a cellmembrane.

Within preferred embodiments of the invention the isolatedpolynucleotides will hybridize to similar sized regions of the DNA ofSEQ ID NO:1, or a sequence complementary thereto, under stringentconditions. In general, stringent conditions are selected to be about 5°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Typicalstringent conditions are those in which the salt concentration is about0.02 M or less at pH 7 and the temperature is at least about 60° C. Aspreviously noted, the isolated polynucleotides of the present inventioninclude DNA and RNA. Methods for isolating DNA and RNA are well known inthe art. Total RNA can be prepared using guanidine HCl extractionfollowed by isolation by centrifugation in a CsCl gradient [Chirgwin etal., Biochemistry 18:52-94 (1979)]. Poly (A)⁺ RNA is prepared from totalRNA using the method of Aviv and Leder, Proc. Natl. Acad. Sci. USA69:1408-1412 (1972). Complementary DNA (cDNA) is prepared from poly(A)⁺RNA using known methods. Polynucleotides encoding Zcyto7 polypeptidesare then identified and isolated by, for example, hybridization or PCR.

Additionally, the polynucleotides of the present invention can besynthesized using a DNA synthesizer. Currently the method of choice isthe phosphoramidite method. If chemically synthesized double strandedDNA is required for an application such as the synthesis of a gene or agene fragment, then each complementary strand is made separately. Theproduction of short genes (60 to 80 bp) is technically straightforwardand can be accomplished by synthesizing the complementary strands andthen annealing them. For the production of longer genes (>300 bp),however, special strategies must be invoked, because the couplingefficiency of each cycle during chemical DNA synthesis is seldom 100%.To overcome this problem, synthetic genes (double-stranded) areassembled in modular form from single-stranded fragments that are from20 to 100 nucleotides in length. In addition to the protein codingsequence, synthetic genes can be designed with terminal sequences thatfacilitate insertion into a restriction endonuclease sites of a cloningvector and other sequences should also be added that contain signals forthe proper initiation and termination of transcription and translation.

See Glick, Bernard R. and Jack J. Pasternak, Molecular Biotechnology,Principles & Applications of Recombinant DNA, (ASM Press, Washington,D.C. 1994), Itakura, K. et al. Synthesis and use of syntheticoligonucleotides. Annu. Rev. Biochem. 53: 323-356 (1984), and Climie, S.et al. Chemical synthesis of the thymidylate synthase gene. Proc. Natl.Acad. Sci. USA 87:633-637 (1990).

Those skilled in the art will recognize that the sequences disclosed inSEQ ID NOS:1 and 2 represent a single allele of the human. There are anumber of naturally occurring mature N-terminal variants having theleader sequence cleaved at differing positions. They include thesequences defined by SEQ ID NOs 14, 36, 37 and 38. Allelic variants ofthese sequences can be cloned by probing cDNA or genomic libraries fromdifferent individuals according to standard procedures. Examples ofvariants of human Zcyto7 are represented by the polypeptides of SEQ IDNOs: 15-25.

The murine Zcyto7 cDNA and protein are disclosed by SEQ ID NOs: 11 and12. The mature Zcyto7 polypeptide is defined by SEQ ID NOs: 39 and 40.

The present invention further provides counterpart proteins andpolynucleotides from other species (“species orthologs”). Of particularinterest are Zcyto7 polypeptides from other mammalian species, includingmurine, porcine, ovine, bovine, canine, feline, equine, and otherprimates. Species orthologs of the human Zcyto7 protein can be clonedusing information and compositions provided by the present invention incombination with conventional cloning techniques. For example, a cDNAcan be cloned using mRNA obtained from a tissue or cell type thatexpresses the protein. Suitable sources of mRNA can be identified byprobing Northern blots with probes designed from the sequences disclosedherein. A library is then prepared from mRNA of a positive tissue orcell line. A protein-encoding cDNA can then be isolated by a variety ofmethods, such as by probing with a complete or partial human or mousecDNA or with one or more sets of degenerate probes based on thedisclosed sequences. A cDNA can also be cloned using the polymerasechain reaction, or PCR (Mullis, U.S. Pat. No. 4,683,202), using primersdesigned from the sequences disclosed herein. Within an additionalmethod, the cDNA library can be used to transform or transfect hostcells, and expression of the cDNA of interest can be detected with anantibody to the protein. Similar techniques can also be applied to theisolation of genomic clones. As used and claimed the language “anisolated polynucleotide which encodes a polypeptide, said polynucleotidebeing defined by SEQ ID NO: 2” includes all allelic variants and speciesorthologs of the polypeptide of SEQ ID NO:2.

The present invention also provides isolated protein polypeptides thatare substantially homologous to the protein polypeptides of SEQ ID NO: 2and its species orthologs. By “isolated” is meant a protein orpolypeptide that is found in a condition other than its nativeenvironment, such as apart from blood and animal tissue. In a preferredform, the isolated polypeptide is substantially free of otherpolypeptides, particularly other polypeptides of animal origin. It ispreferred to provide the polypeptides in a highly purified form, i.e.greater than 95% pure, more preferably greater than 99% pure. The term“substantially homologous” is used herein to denote polypeptides having50%, preferably 60%, more preferably at least 80%, sequence identity tothe sequence shown in SEQ ID NO:2, or its species orthologs. Suchpolypeptides will more preferably be at least 90% identical, and mostpreferably 95% or more identical to SEQ ID NO:2, or its speciesorthologs. Percent sequence identity is determined by conventionalmethods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-616(1986) and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA89:10915-10919 (1992). Briefly, two amino acid sequences are aligned tooptimize the alignment scores using a gap opening penalty of 10, a gapextension penalty of 1, and the “blossom 62” scoring matrix of Henikoffand Henikoff (ibid.) as shown in Table 2 (amino acids are indicated bythe standard one-letter codes). The percent identity is then calculatedas:$\frac{{Total}\quad{number}\quad{of}\quad{identical}\quad{matches}}{\begin{matrix}{\left\lbrack {{length}\quad{of}\quad{the}\quad{longer}\quad{sequence}\quad{plus}\quad{the}} \right.} \\{{number}\quad{of}\quad{gaps}\quad{introduced}\quad{into}\quad{the}\quad{longer}} \\\left. {{sequence}\quad{in}\quad{order}\quad{to}\quad{align}\quad{the}\quad{two}\quad{sequences}} \right\rbrack\end{matrix}} \times 100$ TABLE 2 A R N D C Q E G H I L K M F P S T W YV A 4 R −1 5 N −2 0 6 D −2 −2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 00 2 −4 2 5 G 0 −2 0 −1 −3 −2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3−1 −3 −3 −4 −3 4 L −1 −2 −3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2−1 −3 −2 5 M −1 −1 −2 −3 −1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3−3 −1 0 0 −3 0 6 P −1 −2 −2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 10 −1 0 0 0 −1 −2 −2 0 −1 −2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1−2 −1 1 5 W −3 −3 −4 −4 −2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2−2 −3 −2 −1 −2 −3 2 −1 −1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3−3 3 1 −2 1 −1 −2 −2 0 −3 −1 4

Sequence identity of polynucleotide molecules is determined by similarmethods using a ratio as disclosed above.

Substantially homologous proteins and polypeptides are characterized ashaving one or more amino acid substitutions, deletions or additions.These changes are preferably of a minor nature, that is conservativeamino acid substitutions (see Table 3) and other substitutions that donot significantly affect the folding or activity of the protein orpolypeptide; small deletions, typically of one to about 30 amino acids;and small amino- or carboxyl-terminal extensions, such as anamino-terminal methionine residue, a small linker peptide of up to about20-25 residues, or a small extension that facilitates purification (anaffinity tag), such as a poly-histidine tract, protein A [Nilsson etal., EMBO J. 4:1075 (1985); Nilsson et al., Methods Enzymol. 198:3,(1991)], glutathione S transferase [Smith and Johnson, Gene 67:31,(1988)], or other antigenic epitope or binding domain. See, in generalFord et al., Protein Expression and Purification 2: 95-107 (1991. DNAsencoding affinity tags are available from commercial suppliers (e.g.,Pharmacia Biotech, Piscataway, N.J.). TABLE 3 Conservative amino acidsubstitutions Basic: arginine lysine histidine Acidic: glutamic acidaspartic acid Polar: glutamine asparagine Hydrophobic: leucineisoleucine valine Aromatic: phenylalanine tryptophan tyrosine Small:glycine alanine serine threonine methionine

Essential amino acids in the polypeptides of the present invention canbe identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis [Cunninghamand Wells, Science 244: 1081-1085 (1989); Bass et al., Proc. Natl. Acad.Sci. USA 88:4498-4502 (1991)]. In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for biological activity (e.g.,ligand binding and signal transduction) to identify amino acid residuesthat are critical to the activity of the molecule. Sites ofligand-protein interaction can also be determined by analysis of crystalstructure as determined by such techniques as nuclear magneticresonance, crystallography or photoaffinity labeling. See, for example,de Vos et al., Science 255:306-312 (1992); Smith et al., J. Mol. Biol.224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64 (1992). Theidentities of essential amino acids can also be inferred from analysisof homologies with related proteins.

Multiple amino acid substitutions can be made and tested using knownmethods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer, Science 241:53-57 (1988) or Bowie and Sauer,Proc. Natl. Acad. Sci. USA 86:2152-2156 (1989). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display, e.g., Lowman et al., Biochem. 30:10832-10837 (1991);Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO92/06204) and region-directed mutagenesis, Derbyshire et al., Gene46:145 (1986); Ner et al., DNA 7:127 (1988).

Mutagenesis methods as disclosed above can be combined withhigh-throughput screening methods to detect activity of cloned,mutagenized proteins in host cells. Preferred assays in this regardinclude cell proliferation assays and biosensor-based ligand-bindingassays, which are described below. Mutagenized DNA molecules that encodeactive proteins or portions thereof (e.g., ligand-binding fragments) canbe recovered from the host cells and rapidly sequenced using modernequipment. These methods allow the rapid determination of the importanceof individual amino acid residues in a polypeptide of interest, and canbe applied to polypeptides of unknown structure.

Using the methods discussed above, one of ordinary skill in the art canprepare a variety of polypeptides that are substantially homologous toSEQ ID NO:2 or allelic variants thereof and retain the properties of thewild-type protein. As expressed and claimed herein the language, “apolypeptide as defined by SEQ ID NO: 2” includes all allelic variantsand species orthologs of the polypeptide.

Another embodiment of the present invention provides for a peptide orpolypeptide comprising an epitope-bearing portion of a polypeptide ofthe invention. The epitope of the this polypeptide portion is animmunogenic or antigenic epitope of a polypeptide of the invention. Aregion of a protein to which an antibody can bind is defined as an“antigenic epitope”. See for instance, Geysen, H. M. et al., Proc. Natl.Acad Sci. USA 81:3998-4002 (1984).

As to the selection of peptides or polypeptides bearing an antigenicepitope (i.e., that contain a region of a protein molecule to which anantibody can bind), it is well known in the art that relatively shortsynthetic peptides that mimic part of a protein sequence are routinelycapable of eliciting an antiserum that reacts with the partiallymimicked protein. See Sutcliffe, J. G. et al. Science 219:660-666(1983). Peptides capable of eliciting protein-reactive sera arefrequently represented in the primary sequence of a protein, can becharacterized by a set of simple chemical rules, and are confinedneither to immunodominant regions of intact proteins (i.e., immunogenicepitopes) nor to the amino or carboxyl terminals. Peptides that areextremely hydrophobic and those of six or fewer residues generally areineffective at inducing antibodies that bind to the mimicked protein;longer soluble peptides, especially those containing proline residues,usually are effective.

Antigenic epitope-bearing peptides and polypeptides of the invention aretherefore useful to raise antibodies, including monoclonal antibodies,that bind specifically to a polypeptide of the invention. Antigenicepitope-bearing peptides and polypeptides of the present inventioncontain a sequence of at least nine, preferably between 15 to about 30amino acids contained within the amino acid sequence of a polypeptide ofthe invention. However, peptides or polypeptides comprising a largerportion of an amino acid sequence of the invention, containing from 30to 50 amino acids, or any length up to and including the entire aminoacid sequence of a polypeptide of the invention, also are useful forinducing antibodies that react with the protein. Preferably, the aminoacid sequence of the epitope-bearing peptide is selected to providesubstantial solubility in aqueous solvents (i.e., the sequence includesrelatively hydrophilic residues and hydrophobic residues are preferablyavoided); and sequences containing proline residues are particularlypreferred. All of the polypeptides shown in the sequence listing containantigenic epitopes to be used according to the present invention,however, specifically designed antigenic epitopes include the peptidesdefined by SEQ ID NOs: 27-35.

Polynucleotides, generally a cDNA sequence, of the present inventionencode the above-described polypeptides. A cDNA sequence which encodes apolypeptide of the present invention is comprised of a series of codons,each amino acid residue of the polypeptide being encoded by a codon andeach codon being comprised of three nucleotides. The amino acid residuesare encoded by their respective codons as follows.

Alanine (Ala) is encoded by GCA, GCC, GCG or GCT;

Cysteine (Cys) is encoded by TGC or TGT;

Aspartic acid (Asp) is encoded by GAC or GAT;

Glutamic acid (Glu) is encoded by GAA or GAG;

Phenylalanine (Phe) is encoded by TTC or TTT;

Glycine (Gly) is encoded by GGA, GGC, GGG or GGT;

Histidine (His) is encoded by CAC or CAT;

Isoleucine (Ile) is encoded by ATA, ATC or ATT;

Lysine (Lys) is encoded by AAA, or AAG;

Leucine (Leu) is encoded by TTA, TTG, CTA, CTC, CTG or CTT;

Methionine (Met) is encoded by ATG;

Asparagine (Asn) is encoded by AAC or AAT;

Proline (Pro) is encoded by CCA, CCC, CCG or CCT;

Glutamine (Gln) is encoded by CAA or CAG;

Arginine (Arg) is encoded by AGA, AGG, CGA, CGC, CGG or CGT;

Serine (Ser) is encoded by AGC, AGT, TCA, TCC, TCG or TCT;

Threonine (Thr) is encoded by ACA, ACC, ACG or ACT;

Valine (Val) is encoded by GTA, GTC, GTG or GTT;

Tryptophan (Trp) is encoded by TGG; and

Tyrosine (Tyr) is encoded by TAC or TAT.

It is to be recognized that according to the present invention, when acDNA is claimed as described above, it is understood that what isclaimed are both the sense strand, the anti-sense strand, and the DNA asdouble-stranded having both the sense and anti-sense strand annealedtogether by their respective hydrogen bonds. Also claimed is themessenger RNA (mRNA) which encodes the polypeptides of the presentinvention, and which mRNA is encoded by the above-described cDNA. Amessenger RNA (mRNA) will encode a polypeptide using the same codons asthose defined above, with the exception that each thymine nucleotide (T)is replaced by a uracil nucleotide (U).

The protein polypeptides of the present invention, including full-lengthproteins, protein fragments (e.g. receptor-binding fragments), andfusion polypeptides can be produced in genetically engineered host cellsaccording to conventional techniques. Suitable host cells are those celltypes that can be transformed or transfected with exogenous DNA andgrown in culture, and include bacteria, fungal cells, and culturedhigher eukaryotic cells. Eukaryotic cells, particularly cultured cellsof multicellular organisms, are preferred. Techniques for manipulatingcloned DNA molecules and introducing exogenous DNA into a variety ofhost cells are disclosed by Sambrook et al., Molecular Cloning: ALaboratory Manual, (2nd ed.) (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989), and Ausubel et al., ibid.

In general, a DNA sequence encoding a Zcyto7 polypeptide is operablylinked to other genetic elements required for its expression, generallyincluding a transcription promoter and terminator, within an expressionvector. The vector will also commonly contain one or more selectablemarkers and one or more origins of replication, although those skilledin the art will recognize that within certain systems selectable markersmay be provided on separate vectors, and replication of the exogenousDNA may be provided by integration into the host cell genome. Selectionof promoters, terminators, selectable markers, vectors and otherelements is a matter of routine design within the level of ordinaryskill in the art. Many such elements are described in the literature andare available through commercial suppliers.

To direct a Zcyto7 polypeptide into the secretory pathway of a hostcell, a secretory signal sequence (also known as a leader sequence,prepro sequence or pre sequence) is provided in the expression vector.The secretory signal sequence may be that of the protein, or may bederived from another secreted protein (e.g., t-PA) or synthesized denovo. The secretory signal sequence is joined to the Zcyto7 DNA sequencein the correct reading frame. Secretory signal sequences are commonlypositioned 5′ to the DNA sequence encoding the polypeptide of interest,although certain signal sequences may be positioned elsewhere in the DNAsequence of interest (see, e.g., Welch et al., U.S. Pat. No. 5,037,743;Holland et al., U.S. Pat. No. 5,143,830).

Cultured mammalian cells are preferred hosts within the presentinvention. Methods for introducing exogenous DNA into mammalian hostcells include calcium phosphate-mediated transfection, Wigler et al.,Cell 14:725 (1978); Corsaro and Pearson, Somatic Cell Genetics 7:603(1981): Graham and Van der Eb, Virology 52:456 (1973), electroporation,Neumann et al., EMBO J. 1:841-845 (1982), DEAE-dextran mediatedtransfection, Ausubel et al., eds., Current Protocols in MolecularBiology (John Wiley and Sons, Inc., NY, 1987), and liposome-mediatedtransfection (Hawley-Nelson et al., Focus 15:73 (1993); Ciccarone etal., Focus 15:80 (1993). The production of recombinant polypeptides incultured mammalian cells is disclosed, for example, by Levinson et al.,U.S. Pat. No. 4,713,339; Hagen et al., U.S. Pat. No. 4,784,950; Palmiteret al., U.S. Pat. No. 4,579,821; and Ringold, U.S. Pat. No. 4,656,134.Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650),COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No.CRL 10314), 293 [ATCC No. CRL 1573; Graham et al., J. Gen. Virol.36:59-72 (1977)] and Chinese hamster ovary (e.g. CHO-K1; ATCC No. CCL61) cell lines. Additional suitable cell lines are known in the art andavailable from public depositories such as the American Type CultureCollection, Rockville, Md. In general, strong transcription promotersare preferred, such as promoters from SV-40 or cytomegalovirus. See,e.g., U.S. Pat. No. 4,956,288. Other suitable promoters include thosefrom metallothionein genes (U.S. Pat. Nos. 4,579,821 and 4,601,978) andthe adenovirus major late promoter.

Drug selection is generally used to select for cultured mammalian cellsinto which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Apreferred selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems mayalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.A preferred amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g. hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used.

Other higher eukaryotic cells can also be used as hosts, includinginsect cells, plant cells and avian cells. Transformation of insectcells and production of foreign polypeptides therein is disclosed byGuarino et al., U.S. Pat. No. 5,162,222; Bang et al., U.S. Pat. No.4,775,624; and WIPO publication WO 94/06463. The use of Agrobacteriumrhizogenes as a vector for expressing genes in plant cells has beenreviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58 (1987).

Fungal cells, including yeast cells, and particularly cells of the genusSaccharomyces, can also be used within the present invention, such asfor producing protein fragments or polypeptide fusions. Methods fortransforming yeast cells with exogenous DNA and producing recombinantpolypeptides therefrom are disclosed by, for example, Kawasaki, U.S.Pat. No. 4,599,311; Kawasaki et al., U.S. Pat. No. 4,931,373; Brake,U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat. No. 5,037,743; andMurray et al., U.S. Pat. No. 4,845,075. Transformed cells are selectedby phenotype determined by the selectable marker, commonly drugresistance or the ability to grow in the absence of a particularnutrient (e.g., leucine). A preferred vector system for use in yeast isthe POT1 vector system disclosed by Kawasaki et al., U.S. Pat. No.4,931,373, which allows transformed cells to be selected by growth inglucose-containing media. Suitable promoters and terminators for use inyeast include those from glycolytic enzyme genes (see, e.g., Kawasaki,U.S. Pat. No. 4,599,311; Kingsman et al., U.S. Pat. No. 4,615,974; andBitter, U.S. Pat. No. 4,977,092) and alcohol dehydrogenase genes. Seealso U.S. Pat. Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454.Transformation systems for other yeasts, including Hansenula polymorpha,Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis,Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichiaguillermondii and Candida maltosa are known in the art. See, forexample, Gleeson et al., J. Gen. Microbiol. 132:3459-3465 (1986) andCregg, U.S. Pat. No. 4,882,279. Aspergillus cells may be utilizedaccording to the methods of McKnight et al., U.S. Pat. No. 4,935,349.Methods for transforming Acremonium chrysogenum are disclosed by Suminoet al., U.S. Pat. No. 5,162,228. Methods for transforming Neurospora aredisclosed by Lambowitz, U.S. Pat. No. 4,486,533.

Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell.

Within one aspect of the present invention, a novel protein is producedby a cultured cell, and the cell is used to screen for a receptor orreceptors for the protein, including the natural receptor, as well asagonists and antagonists of the natural ligand.

Protein Isolation:

Expressed recombinant polypeptides (or chimeric polypeptides) can bepurified using fractionation and/or conventional purification methodsand media. Ammonium sulfate precipitation and acid or chaotropeextraction may be used for fractionation of samples. Exemplarypurification steps may include hydroxyapatite, size exclusion, FPLC andreverse-phase high performance liquid chromatography. Suitable anionexchange media include derivatized dextrans, agarose, cellulose,polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Qderivatives are preferred, with DEAE Fast-Flow Sepharose (Pharmacia,Piscataway, N.J.) being particularly preferred. Exemplarychromatographic media include those media derivatized with phenyl,butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia),Toyopearl butyl 650 (Toso Haas, Montgomeryville, Pa.), Octyl-Sepharose(Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG71 (Toso Haas) and the like. Suitable solid supports include glassbeads, silica-based resins, cellulosic resins, agarose beads,cross-linked agarose beads, polystyrene beads, cross-linkedpolyacrylamide resins and the like that are insoluble under theconditions in which they are to be used. These supports may be modifiedwith reactive groups that allow attachment of proteins by amino groups,carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydratemoieties. Examples of coupling chemistries include cyanogen bromideactivation, N-hydroxysuccinimide activation, epoxide activation,sulfhydryl activation, hydrazide activation, and carboxyl and aminoderivatives for carbodiimide coupling chemistries. These and other solidmedia are well known and widely used in the art, and are available fromcommercial suppliers. Methods for binding receptor polypeptides tosupport media are well known in the art. Selection of a particularmethod is a matter of routine design and is determined in part by theproperties of the chosen support. See, for example, AffinityChromatography: Principles & Methods (Pharmacia LKB Biotechnology,Uppsala, Sweden, 1988).

The polypeptides of the present invention can be isolated byexploitation of their properties. For example, immobilized metal ionadsorption (IMAC) chromatography can be used to purify histidine-richproteins. Briefly, a gel is first charged with divalent metal ions toform a chelate [E. Sulkowski, Trends in Biochem. 3:1-7 (1985)].Histidine-rich proteins will be adsorbed to this matrix with differingaffinities, depending upon the metal ion used, and will be eluted bycompetitive elution, lowering the pH, or use of strong chelating agents.Other methods of purification include purification of glycosylatedproteins by lectin affinity chromatography and ion exchangechromatography [Methods in Enzymol., Vol. 182:529-39, “Guide to ProteinPurification”, M. Deutscher, (ed.), (Acad. Press, San Diego, 1990).Alternatively, a fusion of the polypeptide of interest and an affinitytag (e.g., polyhistidine, maltose-binding protein, an immunoglobulindomain) may be constructed to facilitate purification. Furthermore, tofacilitate purification of the secreted receptor polypeptide, an aminoor carboxyl-terminal extension, such as a poly-histidine tag, substanceP, FLAG® peptide [Hopp et al., Bio/Technology 6:1204-1210 (1988);available from Eastman Kodak Co., New Haven, Conn.), a Glu-Glu affinitytag [Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4 (1985)],or another polypeptide or protein for which an antibody or otherspecific binding agent is available, can be fused to Zyto7 to aid inpurification.

Uses

Northern blot analysis of the expression of Zcyto7 reveals that Zcyto7is specifically expressed in the spinal cord. In situ analysis of thespinal cord reveals that this expression is localized in the neurons anddorsal root ganglia. Therefore, Zcyto7 may play a role in themaintenance of spinal cord involving either glial cells or neurons. Thisindicates that Zcyto7 can be used to treat a variety ofneurodegenerative diseases such as amyotrophic lateral sclerosis (ALS),or dymyelinating diseases including multiple sclerosis. Zcyto7 may alsobe used to treat sensory neuropathis. The tissue specificity of Zcyto7expression suggests that Zcyto7 may be a growth and/or maintenancefactor in the spinal cord.

Zcyto7 gene's location on chromosome 5 indicates that zcyto7 is acytokine which can be used to modulate the activities of cells of theimmune system. Zcyto7 can also be used as a chemoattractant ofneutrophils in the spinal column. This would be useful as ananti-infective for infections in the spinal column. It could also beused to help regulate other cytokines in the spinal cord. Zcyto7 mayalso be administered to treat peripheral neuropathies such asCharcot-Marie-Tooth (CMT) disease which is localized to the samechromosomal region of 5q as Zcyto7.

The fact that Zcyto7 inhibits the growth of BAF-3 and TF-1 cells asshown in examples 11 and 12 below indicates that Zcyto7 can be used totreat autoimmune diseases and possibly such cancers such as leukemias.

The present invention also provides reagents which will find use indiagnostic applications. For example, the Zcyto7 gene is heavilyexpressed in the spinal cord. A probe comprising the Zcyto7 DNA or RNAor a subsequence thereof can be used to determine if the Zcyto7 gene ispresent on chromosome 5 or if a mutation has occurred.

The present invention also provides reagents with significanttherapeutic value. The Zcyto7 polypeptide (naturally occurring orrecombinant), fragments thereof, antibodies and anti-idiotypicantibodies thereto, along with compounds identified as having bindingaffinity to the Zcyto7 polypeptide, should be useful in the treatment ofconditions associated with abnormal physiology or development, includingabnormal proliferation, e.g., cancerous conditions, or degenerativeconditions. For example, a disease or disorder associated with abnormalexpression or abnormal signaling by a Zcyto7 polypeptide should be alikely target for an agonist or antagonist of the Zcyto7 polypeptide.

In particular, Zcyto7 can be used to treat inflammation. Inflammation isa result of an immune response to an infection or as an autoimmuneresponse to a self-antigen.

Treatment dosages should be titrated to optimize safety and efficacy.Methods for administration include intravenous, peritoneal,intramuscular, subdural, into the spinal fluid or transdermaladministration. Pharmaceutically acceptable carriers will include water,saline, buffers to name just a few. Dosage ranges would ordinarily beexpected from 0.1 μg to 1 mg per kilogram of body weight per day.Preferably, 1 μg to 100 μg per day. However, the doses by be higher orlower as can be determined by a medical doctor with ordinary skill inthe art. For a complete discussion of drug formulations and dosageranges see Remington's Pharmaceutical Sciences, 17^(th) Ed., (MackPublishing Co., Easton, Pa., 1990), and Goodman and Gilman's: ThePharmacological Bases of Therapeutics, 9^(th) Ed. (Pergamon Press 1996).

Use of Zcyto7 to Promote Bone and Cartilage Growth

It has been discovered that Zcyto7 stimulates the proliferation of bothchondrocytes and osteoblasts as is shown below in Examples 7 and 9respectively. In addition, Zcyto7 also stimulates the steady state levelof glycosaminoglycan present in chondrocyte cultures as shown in Example8. Thus Zcyto7 can be used to stimulate both bone and cartilage growthin a variety of different therapeutic settings.

Zcyto7 can be implanted in a mammalian body so that the zcyto7 is incontact with osteoblasts such that osteoblast proliferation occurs andbone growth is stimulated. For example, zcyto7 can be placed in a matrix[with or without a bone morphogenic protein (BMP)]. The BMP induces themigration of mesenchymal osteoblast precursors to the site and furtherinduces differentiation of the mesenchymal cells into osteoblast. Zcyto7will then stimulate the further proliferation of the osteoblasts. Asuitable matrix is made up of particles of porous materials. The poresmust be of a dimension to permit progenitor cell migration andsubsequent differentiation and proliferation. An ideal particle sizeshould be in the range of 70-850 mm, preferably 150-420 mm. The matrixcontaining the zcyto7 can be molded into a shape encompassing a bonedefect. Examples of matrix materials are particulate, demineralized,guanidine extracted, species-specific bone. Other potentially usefulmatrix materials include collagen, homopolymers and copolymers ofglycolic acid and lactic acid, hydroxyapatite, tricalcium phosphate andother calcium phosphates. Zcyto7 can be applied into a matrix at asufficient concentration to promote the proliferation of osteoblasts,preferably at a concentration of at least 1 μg/ml of matrix. A solutionof zcyto7 can also be injected directly into the site of a bone fractureor defect including areas of bone degeneration to expedite healing ofthe fracture or defect site. Examples of BMPs and the use of matrices toproduce are disclosed in PCT application publication number WO 92/07073,publication No. WO 91/05802, U.S. Pat. No. 5,645,591 and U.S. Pat. No.5,108,753.

Zcyto7 can be further used to treat osteoporosis by administering atherapeutically effective amount of zcyto7 to an individual. A preferreddosage would be 1 μg of zcyto7 per kilogram of body weight per day.

As stated above, it has also been determined that zcyto7 can be used topromote the production of cartilage through its ability to stimulate theproliferation of chondrocytes. Zcyto7 can be injected directly into thesite where cartilage is to be grown. For example, zcyto7 can be injecteddirectly in joints which have been afflicted with osteoarthritis orother injured joints in which the cartilage has been worn down. Anexample of a case in which additional cartilage needs to be grown isshoulders and knees of injured athletes.

Cartilage can also be grown by first removing chondrocytes from anindividual, culturing the chondrocytes with zcyto7 so that theyproliferate and reimplanting the chondrocytes back into the individualwhere cartilage needs to be produced.

Zcyto7 can also be used to stimulate the regeneration of dentin or bonewhich has been lost due to periodontal disease. To do this, thesurrounding tissue should be thoroughly cleaned and a solution of Zcto7be administered, preferably by injection, into the site in which dentinregeneration is desired.

Antibodies to the zcyto7 polypeptide can be purified and thenadministered to a patient. These reagents can be combined fortherapeutic use with additional active or inert ingredients, e.g., inpharmaceutically acceptable carriers or diluents along withphysiologically innocuous stabilizers and excipients. These combinationscan be sterile filtered and placed into dosage forms as bylyophilization in dosage vials or storage in stabilized aqueouspreparations. This invention also contemplates use of antibodies,binding fragments thereof or single-chain antibodies of the antibodiesincluding forms which are not complement binding.

The quantities of reagents necessary for effective therapy will dependupon many different factors, including means of administration, targetsite, physiological state of the patient, and other medicationsadministered. Thus, treatment dosages should be titrated to optimizesafety and efficacy. Typically, dosages used in vitro may provide usefulguidance in the amounts useful for in vivo administration of thesereagents. Animal testing of effective doses for treatment of particulardisorders will provide further predictive indication of human dosage.Methods for administration include oral, intravenous, peritoneal,intramuscular, or transdermal administration. Pharmaceuticallyacceptable carriers will include water, saline, buffers to name just afew. Dosage ranges would ordinarily be expected from 1 μg to 1000 μg perkilogram of body weight per day. However, the doses may be higher orlower as can be determined by a medical doctor with ordinary skill inthe art. For a complete discussion of drug formulations and dosageranges see Remington's Pharmaceutical Sciences, 17^(th) Ed., (MackPublishing Co., Easton, Pa., 1990), and Goodman and Gilman's: ThePharmacological Bases of Therapeutics, 9^(th) Ed. (Pergamon Press 1996).

Nucleic Acid-Based Therapeutic Treatment

If a mammal has a mutated or lacks a Zcyto7 gene, the Zcyto7 gene can beintroduced into the cells of the mammal. In one embodiment, a geneencoding a Zcyto7 polypeptide is introduced in vivo in a viral vector.Such vectors include an attenuated or defective DNA virus, such as butnot limited to herpes simplex virus (HSV), papillomavirus, Epstein Barrvirus (EBV), adenovirus, adeno-associated virus (AAV), and the like.Defective viruses, which entirely or almost entirely lack viral genes,are preferred. A defective virus is not infective after introductioninto a cell. Use of defective viral vectors allows for administration tocells in a specific, localized area, without concern that the vector caninfect other cells. Examples of particular vectors include, but are notlimited to, a defective herpes virus 1 (HSV1) vector [Kaplitt et al.,Molec. Cell. Neurosci., 2:320-330 (1991)], an attenuated adenovirusvector, such as the vector described by Stratford-Perricaudet et al., J.Clin. Invest., 90:626-630 (1992), and a defective adeno-associated virusvector [Samulski et al., J. Virol., 61:3096-3101 (1987); Samulski et al.J. Virol., 63:3822-3828 (1989)].

In another embodiment, the gene can be introduced in a retroviralvector, e.g., as described in Anderson et al., U.S. Pat. No. 5,399,346;Mann et al., Cell, 33:153 (1983); Temin et al., U.S. Pat. No. 4,650,764;Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., J. Virol.,62:1120 (1988); Temin et al., U.S. Pat. No. 5,124,263; InternationalPatent Publication No. WO 95/07358, published Mar. 16, 1995 by Doughertyet al.; and Blood, 82:845 (1993).

Alternatively, the vector can be introduced by lipofection in vivo usingliposomes. Synthetic cationic lipids can be used to prepare liposomesfor in vivo transfection of a gene encoding a marker [Felgner et al.,Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987); see Mackey et al.,Proc. Natl. Acad. Sci. USA, 85:8027-8031 (1988)]. The use of lipofectionto introduce exogenous genes into specific organs in vivo has certainpractical advantages. Molecular targeting of liposomes to specific cellsrepresents one area of benefit. It is clear that directing transfectionto particular cells represents one area of benefit. It is clear thatdirecting transfection to particular cell types would be particularlyadvantageous in a tissue with cellular heterogeneity, such as thepancreas, liver, kidney, and brain. Lipids may be chemically coupled toother molecules for the purpose of targeting. Targeted peptides, e.g.,hormones or neurotransmitters, and proteins such as antibodies, ornon-peptide molecules could be coupled to liposomes chemically.

It is possible to remove the cells from the body and introduce thevector as a naked DNA plasmid and then re-implant the transformed cellsinto the body. Naked DNA vector for gene therapy can be introduced intothe desired host cells by methods known in the art, e.g., transfection,electroporation, microinjection, transduction, cell fusion, DEAEdextran, calcium phosphate precipitation, use of a gene gun or use of aDNA vector transporter [see, e.g., Wu et al., J. Biol. Chem.,267:963-967 (1992); Wu et al., J. Biol. Chem., 263:14621-14624 (1988)].

Zcyto7 polypeptides can also be used to prepare antibodies thatspecifically bind to Zcyto7 polypeptides. These antibodies can then beused to manufacture anti-idiotypic antibodies. As used herein, the term“antibodies” includes polyclonal antibodies, monoclonal antibodies,antigen-binding fragments thereof such as F(ab′)₂ and Fab fragments, andthe like, including genetically engineered antibodies. Antibodies aredefined to be specifically binding if they bind to a Zcyto7 polypeptidewith a K_(a) of greater than or equal to 10⁷/M. The affinity of amonoclonal antibody can be readily determined by one of ordinary skillin the art (see, for example, Scatchard, ibid.).

Methods for preparing polyclonal and monoclonal antibodies are wellknown in the art (see for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, (Second Edition) (Cold Spring Harbor, N.Y., 1989);and Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniquesand Applications (CRC Press, Inc., Boca Raton, Fla., 1982). As would beevident to one of ordinary skill in the art, polyclonal antibodies canbe generated from a variety of warm-blooded animals such as horses,cows, goats, sheep, dogs, chickens, rabbits, mice, and rats. Theimmunogenicity of a Zcyto7 polypeptide may be increased through the useof an adjuvant such as Freund's complete or incomplete adjuvant. Avariety of assays known to those skilled in the art can be utilized todetect antibodies which specifically bind to Zcyto7 polypeptides.Exemplary assays are described in detail in Antibodies: A LaboratoryManual, Harlow and Lane (Eds.), (Cold Spring Harbor Laboratory Press,1988). Representative examples of such assays include: concurrentimmunoelectrophoresis, radio-immunoassays, radio-immunoprecipitations,enzyme-linked immunosorbent assays (ELISA), dot blot assays, inhibitionor competition assays, and sandwich assays.

As would be evident to one of ordinary skill in the art, polyclonalantibodies can be generated by inoculating a variety of warm-bloodedanimals such as horses, cows, goats, sheep, dogs, chickens, rabbits,mice, hamsters, guinea pigs and rats with a Zcyto7 polypeptide or afragment thereof. The immunogenicity of a Zcyto7 polypeptide may beincreased through the use of an adjuvant, such as alum (aluminumhydroxide) or Freund's complete or incomplete adjuvant. Polypeptidesuseful for immunization also include fusion polypeptides, such asfusions of Zcyto7 or a portion thereof with an immunoglobulinpolypeptide or with maltose binding protein. The polypeptide immunogenmay be a full-length molecule or a portion thereof. If the polypeptideportion is “hapten-like”, such portion may be advantageously joined orlinked to a macromolecular carrier (such as keyhole limpet hemocyanin(KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.

As used herein, the term “antibodies” includes polyclonal antibodies,affinity-purified polyclonal antibodies, monoclonal antibodies, andantigen-binding fragments, such as F(ab′)₂ and Fab proteolyticfragments. Genetically engineered intact antibodies or fragments, suchas chimeric antibodies, F_(V) fragments, single chain antibodies and thelike, as well as synthetic antigen-binding peptides and polypeptides,are also included. Non-human antibodies may be humanized by graftingnon-human CDRs onto human framework and constant regions, or byincorporating the entire non-human variable domains (optionally“cloaking” them with a human-like surface by replacement of exposedresidues, wherein the result is a “veneered” antibody). In someinstances, humanized antibodies may retain non-human residues within thehuman variable region framework domains to enhance proper bindingcharacteristics. Through humanizing antibodies, biological half-life maybe increased, and the potential for adverse immune reactions uponadministration to humans is reduced.

Alternative techniques for generating or selecting antibodies usefulherein include in vitro exposure of lymphocytes to Zcyto7 protein orpeptide, and selection of antibody display libraries in phage or similarvectors (for instance, through use of immobilized or labeled Zcyto7protein or peptide). Genes encoding polypeptides having potential Zcyto7polypeptide binding domains can be obtained by screening random peptidelibraries displayed on phage (phage display) or on bacteria, such as E.coli. Nucleotide sequences encoding the polypeptides can be obtained ina number of ways, such as through random mutagenesis and randompolynucleotide synthesis. These random peptide display libraries can beused to screen for peptides which interact with a known target which canbe a protein or polypeptide, such as a ligand or receptor, a biologicalor synthetic macromolecule, or organic or inorganic substances.Techniques for creating and screening such random peptide displaylibraries are known in the art (Ladner et al., U.S. Pat. No. 5,223,409;Ladner et al., U.S. Pat. No. 4,946,778; Ladner et al., U.S. Pat. No.5,403,484 and Ladner et al., U.S. Pat. No. 5,571,698) and random peptidedisplay libraries and kits for screening such libraries are availablecommercially, for instance from Clontech (Palo Alto, Calif.), InvitrogenInc. (San Diego, Calif.), New England Biolabs, Inc. (Beverly, Mass.) andPharmacia LKB Biotechnology Inc. (Piscataway, N.J.). Random peptidedisplay libraries can be screened using the Zcyto7 sequences disclosedherein to identify proteins which bind to Zcyto7. These “bindingproteins” which interact with Zcyto7 polypeptides can be used fortagging cells; for isolating homolog polypeptides by affinitypurification; they can be directly or indirectly conjugated to drugs,toxins, radionuclides and the like. These binding proteins can also beused in analytical methods such as for screening expression librariesand neutralizing activity. The binding proteins can also be used fordiagnostic assays for determining circulating levels of polypeptides;for detecting or quantitating soluble polypeptides as marker ofunderlying pathology or disease. These binding proteins can also act asZcyto7 “antagonists” to block Zcyto7 binding and signal transduction invitro and in vivo.

Antibodies can also be generated gene therapy. The animal isadministered the DNA or RNA which encodes Zcyto7 or an immunogenicfragment thereof so that cells of the animals are transfected with thenucleic acid and express the protein which in turn elicits animmunogenic response. Antibodies which then are produced by the animalare isolated in the form of polyclonal or monoclonal antibodies.

Antibodies to Zcyto7 may be used for tagging cells that express theprotein, for affinity purification, within diagnostic assays fordetermining circulating levels of soluble protein polypeptides, and asantagonists to block ligand binding and signal transduction in vitro andin vivo.

Radiation hybrid mapping is a somatic cell genetic technique developedfor constructing high-resolution, contiguous maps of mammalianchromosomes [Cox et al., Science 250:245-250 (1990)]. Partial or fullknowledge of a gene's sequence allows the designing of PCR primerssuitable for use with chromosomal radiation hybrid mapping panels.Commercially available radiation hybrid mapping panels which cover theentire human genome, such as the Stanford G3 RH Panel and the GeneBridge4 RH Panel (Research Genetics, Inc., Huntsville, Ala.), are available.These panels enable rapid, PCR based, chromosomal localizations andordering of genes, sequence-tagged sites (STSs), and othernonpolymorphic- and polymorphic markers within a region of interest.This includes establishing directly proportional physical distancesbetween newly discovered genes of interest and previously mappedmarkers. The precise knowledge of a gene's position can be useful in anumber of ways including: 1) determining if a sequence is part of anexisting contig and obtaining additional surrounding genetic sequencesin various forms such as YAC-, BAC- or cDNA clones, 2) providing apossible candidate gene for an inheritable disease which shows linkageto the same chromosomal region, and 3) for cross-referencing modelorganisms such as mouse which may be beneficial in helping to determinewhat function a particular gene might have.

The present invention also provides reagents which will find use indiagnostic applications. For example, the Zcyto7 gene has been mapped onchromosome 5q31. A Zcyto7 nucleic acid probe could to used to check forabnormalities on chromosome 5. For example, a probe comprising Zcyto7DNA or RNA or a subsequence thereof can be used to determine if theZcyto7 gene is present on chromosome 5q31 or if a mutation has occurred.Detectable chromosomal aberrations at the Zcyto7 gene locus include butare not limited to aneuploidy, gene copy number changes, insertions,deletions, restriction site changes and rearrangements. Such aberrationscan be detected using polynucleotides of the present invention byemploying molecular genetic techniques, such as restriction fragmentlength polymorphism (RFLP) analysis, short tandem repeat (STR) analysisemploying PCR techniques, and other genetic linkage analysis techniquesknown in the art [Sambrook et al., ibid.; Ausubel, et. al., ibid.;Marian, A. J., Chest, 108: 255-265, (1995)].

Zcyto7 maps at the 5q31 region which is a “gene cluster” which containsa group of cytokines and cytokine receptors. The cytokines clusteredthere include IL-3, IL-4, IL-5, IL-13, GM-CSF, and M-CSF. This resultauthenticates zcyto7 as a cytokine.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLE 1 Cloning of Zcyto7

Zcyto7 was identified from expressed sequence tag defined (EST) 582069(SEQ ID NO: 3) by its homology to Interleukin-17. The EST 582069 cDNAclone was obtained from a human fetal heart cDNA library from the IMAGEconsortium, Lawrence Livermore National Laboratory through GenomeSystems, Inc. The cDNA was supplied as an agar stab containing E. colitransfected with the plasmid having the cDNA of interest. The plasmidcontaining the cDNA was streaked out on an LB 100 μg/ml ampicillin and100 μg/ml methicillin plate. The cDNA insert was sequenced. The insertwas determined to be 717 base pairs long with a 180 amino acid openreading frame and a putative 20 amino acid signal peptide.

EXAMPLE 2 Northern Blot Analysis

Human multiple tissue blots 1,2,3 (Clontech)were probed to determine thetissue distribution of Zcyto7. A EcoRI/NotI fragment containing theentire Zcyto7 coding region was generated from the EST582069 clone andused for the probe. A plasmid prep of EST582069 was prepared from a 5 mlLB 100 μg/ml ampicillin overnight culture at 37° using the QIAprep SpinMiniprep Kit (Qiagen). 12 μl out of 100 μl were digested with 5 μl of Hbuffer (Boehringer Mannheim), 12.5 units of EcoRI (Gibco BRL) and 12.5units Not1 (New England Biolabs) in a 50 μl reaction at 37° C. for 2hours. The digest was electrophoresed on a 0.7% TBE agarose gel and thefragment was cut out. To obtain additional material the digest wasrepeated under the same conditions as above except 24 μl of EST582069was used in the second digest. The second digest was electrophoresed ona 0.7% TBE agarose gel and the fragment was cut out. The DNA wasextracted from both gel slabs with a QIAquick Gel Extraction Kit(Qiagen). 135 ng of this DNA was labeled with P³² using the MultiprimeDNA Labeling System (Amersham) and unincorporated radioactivity wasremoved with a NucTrap Probe Purification Column (Stratagene). Multipletissue northerns and a human RNA master blot were prehybridized 3 hourswith 10 ml ExpressHyb Solution (Clontech) containing 1 mg salmon spermDNA which was boiled 5 minutes and then iced 1 minute and added to 10 mlof ExpressHyb Solution, mixed and added to blots. Hybridization wascarried out overnight at 65° C. Initial wash conditions were as follows:2×SSC, 0.05% SDS RT for 40 minutes with several changes of solution then0.1×SSC, 0.1% SDS at 50° C. for 40 minutes, 1 solution change. Blotswere than exposed to film a −80° C. for 5 hours. There was crosshybridization/background so blots were further washed at 55° C. then 65°C. with 0.1%×SSC, 0.1% SDS for 1 hour each. Spinal cord showed very highexpression of Zcyto7 mRNA and trachea showed weak expression of mRNA.The transcript size was approximately 0.75 kb.

EXAMPLE 3 Chromosomal Assignment and Placement of Zcyto7

Zcyto7 was mapped to chromosome 5 using the commercially available“GeneBridge 4 Radiation Hybrid Panel” (Research Genetics, Inc.,Huntsville, Ala.). The GeneBridge 4 Radiation Hybrid Panel containsPCRable DNAs from each of 93 radiation hybrid clones, plus two controlDNAs (the HFL donor and the A23 recipient). A publicly available WWWserver (http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl) allowsmapping relative to the Whitehead Institute/MIT Center for GenomeResearch's radiation hybrid map of the human genome (the “WICGR”radiation hybrid map of the human genome) which was constructed with theGeneBridge 4 Radiation Hybrid Panel.

For the mapping of Zcyto7 with the “GeneBridge 4 RH Panel”, 20

1 reactions were set up in a PCRable 96-well microtiter plate(Stratagene, La Jolla, Calif.) and used in a “RoboCycler Gradient 96”thermal cycler (Stratagene). Each of the 95 PCR reactions consisted of 2

1 10× KlenTaq PCR reaction buffer (CLONTECH Laboratories, Inc., PaloAlto, Calif.), 1.6

1 dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster City, Calif.), 1

1 sense primer SEQ ID NO:4, 1

1 antisense primer SEQ ID NO:5, 2

1 “RediLoad” (Research Genetics, Inc., Huntsville, Ala.), 0.4

1 50× Advantage KlenTaq Polymerase Mix (Clontech Laboratories, Inc.), 25ng of DNA from an individual hybrid clone or control and x

1 ddH₂O for a total volume of 20

1. The reactions were overlaid with an equal amount of mineral oil andsealed. The PCR cycler conditions were as follows: an initial 1 cycle 5minute denaturation at 95° C., 35 cycles of a 1 minute denaturation at95° C., 1 minute annealing at 52° C. and 1 minute extension at 72° C.,followed by a final 1 cycle extension of 7 minutes at 72° C. Thereactions were separated by electrophoresis on a 3% NuSieve GTG agarosegel (FMC Bioproducts, Rockland, Me.).

The results showed that Zcyto7 maps 490.89 cR from the top of the humanchromosome 5 linkage group on the WICGR radiation hybrid map. Relativeto the centromere, its nearest proximal marker was D5S413 and itsnearest distal maker was WI-5208. The use of surrounding markersposition Zcyto7 in the 5q31.3-q32 region on the integrated LDBchromosome 5 map (The Genetic Location Database, University ofSouthhampton, WWW server:http://cedar.genetics.soton.ac.uk/public_html/).

EXAMPLE 4 Construction of Zcyto7 Expression Vectors

Two Zcyto7 construction vectors were made in a FLAG amino acid sequence(SEQ ID NO: 10) was inserted onto the N-terminal or C-terminal ends ofthe Zcyto7 polypeptide. For the construction in which the FLAG aminoacid sequence was attached to the N-terminus of Zcyto7, a 473 bp Zcyto7PCR DNA fragment was generated with 1 μl of a

dilution of the EST582069 plasmid prep of Example 2 and 20 picomoles(pm) of primer SEQ ID NO: 6 and 20 pm primer SEQ ID NO: 7. The PCRreaction was incubated at 94° C. for 1 minute, and then run for 5 cycleseach individual cycle being comprised of 20 seconds at 94° C. and 2minutes at 64° C. This was followed by 22 cycles each cycle beingcomprised of 20 seconds at 94° C. and 2 minutes at 74° C. The reactionwas ended with an incubation for 10 minutes at 74° C. 50 μl of the PCRreaction mixture was digested with 30 units of BamH1 (BoehringerMannheim) and 120 units of Xho1 (Boehringer Mannheim) for 2 hours at 37°C. The digested reaction mixture was electrophoresed on a 1% TBE gel;the DNA band was excised with a razor blade and the DNA was extractedfrom the gel with the Qiaquick<< Gel Extraction Kit (Qiagen). Theexcised DNA was subcloned into plasmid nfpzp9 which had been cut withBam and Xho. Nfpzp9 is a mammalian cell expression vector comprising anexpression cassette containing the mouse metallothionein-1 promoter, asequence encoding the tissue plasminogen activator (TPA) leader, thenthe FLAG peptide (SEQ ID NO:10), then multiple restriction sites. Thesewere followed by the human growth hormone terminator, an E. coli originof replication and a mammalian selectable marker expression unitcontaining the SV40 promoter, enhancer and origin of replication; adihydrofolate reductase gene (DHFR) and the SV40 terminator.

For the construction of the zcyto7 gene in which a C-terminus FLAG wasinserted onto the C-terminus of the zcyto7 polypeptide, a 543 bp zcyto7PCR fragment was generated with 1 μl of

dilution of the EST582069 plasmid preparation described in Example 1 and20 pm each of primers SEQ ID NO: 8 and SEQ ID NO: 9. The PCR reactionwas incubated at 94° C. for 1 minute, then run for 5 cycles, each cyclebeing comprised 20 seconds at 94° C. and 2 minutes at 55° C. This wasfollowed by 22 cycles each cycle comprised of 20 seconds at 94° C. and 2minutes at 74° C. The reaction was ended with a final 10 minuteextension at 74° C. The entire reaction mixture was run on a 1% TBE geland the DNA was cut out with a razor blade and the DNA was extractedusing the QIAQUICK™ gel extraction kit. 20 μl out of the recovered 35 μldigested with 10 units of BamH1 (Boehringer Mannheim) and 10 units ofEcoR1(Gibco BRL) for 2 hours at 37° C. The digested PCR mixture waselectrophoresed on a 1% TBE gel. The DNA band was cut out with a razorblade and the DNA was extracted from the gel using the QIAquick<< GelExtraction Kit (Qiagen). The extracted DNA was subcloned into plasmidcfpzp9 which had been cut with EcoR1 and BamH1. Plasmid cfpzp9 is amammalian expression vector containing an expression cassette having themouse metallothionein-1 promoter, multiple restriction sites forinsertion of coding sequences, a sequence encoding the FLAG peptide, SEQID NO:10, a stop codon, a human growth hormone terminator, an E. coliorigin of replication, a mammalian selectable marker expression unithaving an SV40 promoter, enhancer and origin of replication, a DHFR geneand the SV40 terminator.

Using antibodies to the FLAG polypeptides, one can separate theFLAG-tagged Zcyto7 polypeptides from a cell supernatant liquid.

EXAMPLE 5 Cloning of Murine Zcyto7

Mouse Zcyto7 was identified from EST 660242 SEQ ID NO:14 by its homologyto human Zcyto7. The cDNA clone was obtained from Lawrence LivermoreNational Laboratory through Genome Systems from a murine embryo cDNAlibrary in which the embryos were between 13.5 and 14.5 days old. ThecDNA was supplied as an agar stab containing E. coli transfected withthe plasmid having the cDNA of interest and then streaked out on an LB100 μg/ml ampicillin, 25 μg/ml methicillin plate. The cDNA insert inEST660242 was sequenced. The insert was determined to be 785 base pairswith an open reading frame of 180 amino acids and a putative 20 aminoacid signal peptide. The sequences are defined by SEQ ID NO:11 and SEQID NO:12.

EXAMPLE 6 Tissue Distribution of Murine Zcyto7

Mouse Multiple Tissue Northern Blot (Clontech, Palo Alto, Calif.), mousenorthern dot blot (Clontech), a mouse embryo northern blot, and a mousespinal cord dot blot were probed to determine the tissue distribution ofmurine Zcyto7.

The mouse embryo RNA were isolated from mouse embryos which were 6 tonine days from the date of fertilization using the POLY (A) PURE® mRNAisolation kit (Ambion). 100 mg of each mouse embryo was lysed in 1 ml oflysis buffer, homogenized and processed in batch method according to themanufacturer's protocol. For the northern blot, 2 μg of RNA was loadedon 1.5% agarose, 2.2M formaldehyde gel. The gel was run at 60V for fourhours and 30 minutes. The RNA was transferred overnight onto a Nytranmembrane which had been pre-wetted in 20×SSC. The RNAs were crosslinkedonto the membrane by UV light and baked at 80° C. for 1 hour.

The mouse spinal cord RNA was also prepared with the POLY (A) PURE® mRNAisolation kit (Ambion). The mouse spinal cord dot blot was made byspotting a dot with 1, 2 and 3 μl of RNA at 1 μg RNA/μl concentrationonto Nytran membrane.

A Not1/EcoRI fragment containing the entire Zcyto7 coding region wasgenerated from the clone containing SEQ ID NO: 12 (hereinafter referredto as the SEQ ID NO:12 clone) and was used for the probe. A plasmid prepof the SEQ ID NO:12 clone was prepared from a 5 ml LB 100 μg/mlampicillin overnight culture at 37° C. using the QIAPREP SPIN MINIPREPkit (Qiagen). 4.66 μg were digested with 8 μl of high buffer (BoehringerMannheim), 20 units of Not1 (Biolabs) and 20 units of EcoRI (Gibco BRL)in a 80 μl reaction at 37° C. for 2 hours. The digest waselectrophoresed on a 1.0% TBE gel and the fragment was cut out. The DNAwas extracted from the gel slab with a QIAQUICK® gel extraction kit(Qiagen). 98.8 ng of this fragment was labeled with p³² using theMULTIMPRIME® DNA labeling system (Amersham) and unincorporatedradioactivity was removed with a NUCTRAP® probe purification column(Stratagene).

The two northern preps and the two dot blot preps were prehybridized for3 hours at 65° C. as follows. 1 mg of salmon sperm was boiled 5 minutes,iced 1 minute, mixed with 10 ml of EXPRESSHYB® solution and added to theblots. Hybridization was carried out overnight at 65° C. Initial washconditions were as follows: 2×SSC, 0.1% SDS for 40 minutes at roomtemperature then 0.1×SSC, 0.1% SDS for 40 minutes at 50° C. Blots wereexposed to film overnight at −80° C. The northern blots and the mousedot blot were further washed with 0.1%×SSC, 0.1% SDS at 60° C. to removebackground. The mouse spinal cord dot blot was washed again at higherstringency with 0.1×SSC, 0.1% SDS at 65° C. to confirm the earlierresults.

-   Results: Mouse Zcyto7 expression was seen in the spinal cord,    submaxillary gland and epididymis. Mouse embryo showed expression of    Zcyto7 starting on day 12, peaking at day 16 and ending day 17 from    the date of fertilization. The transcript size was approximately 1    kb.

EXAMPLE 7 Proliferation of Chondrocytes Using Zcyto7

A chondrocyte proliferation assay was done to determine the effect thatZcyto7 would have on chondrocyte proliferation. The assay was done with20% confluent cultures. As a control vehicle, bovine serum albumin wasadded to a culture of chondrocytes instead of Zcyto7. The assay measuredthe 3H-thymidine incorporation of nascent DNA in the chondrocytes, Wahlet al., Mol. Cell. Biol. 8:5016-5025 (1988).

-   Results: A 3.5-9 fold stimulation of primary chondrocyte    proliferation was seen upon exposure of the chondrocyte cultures to    1 μg/ml of zcyto7. Chondrocyte stimulation by zcyto7 was seen with    multiple preparations of the protein and was seen across species    lines. It contrast to this, the control experiment using BSA    resulted in no stimulation of chondrocytes.

EXAMPLE 8 Production of Glycosaminoglycan by Zcyto7-Treated Chondrocytes

A 20% confluent culture of chondrocytes was prepared and zcyto7 wasapplied at a concentration of 1 μg/ml. In a second experiment inaddition to zcyto7, IL-1β was applied to the cell culture. In a controlgroup BSA was added to the chondrocyte culture. The level ofglycosaminoglycan (GAG) production by the chondrocyte culture was thendetermined using a 1,9-dimethylmethlyene blue dye binding assay, Fardaleet al., Biochem. Biophys. Acta 888:173-177 (1987).

-   Results: Chondrocytes which were cultured with zcyto7 showed a 50%    increase in the steady state presence of GAG in the chondrocyte    culture. Moreover, when the chondrocytes were co-cultured with both    zcyto7 and interleukin-1β (IL-1)the GAG production by the    chondrocytes increased 2.5 fold as compared with culturing of the    chondrocytes with either zcyto7 or IL-1β alone. While the cultured    cells to which BSA was added showed no increased production of GAG.

EXAMPLE 9 Osteoblast Stimulation by Zcyto7

The CCC4 cell line is an osteoblast-like cell line derived from p53knockout mice. The CCC4 line was transfected with a plasmid containingan inducible serum response element (SRE) driving the expression ofluciferase. The stimulation of the SRE and thus the expression ofluciferase indicates that the chemical entity is likely to stimulateosteoblasts.

CCC4 cells were cultured in the presence of 1 μg of zcyto7/ml of culturemedium. As a control BSA, fibroblast growth factor (FGF) andplatelet-derived growth factor (PDGF) were each added to differentcultures of CCC4 cells. BSA was a negative control and FGF and PDGF werepositive controls as they are known to promote osteoblast proliferation.Luciferase activity was detected by addition of 40 μl of Promegaluciferase substrate using a 2 second integrated read on LabsystesLUMINOSKAN^(<<).

Results

Zcyto7 as well as, FGF and PDGF stimulate the expression of luciferasein this assay indicating that they stimulate osteoblasts. The BSAvehicle control was negative in this assay.

EXAMPLE 10 Effect of Zcyto7 on the Growth of Fibroblasts

Confluent cultures of human dermal, lung, and fetal lung fibroblastswere inoculated with Zcyto7 to determine the effects of Zcyto7 on thegrowth of fibroblasts. FGF was used as a positive control and BSA as anegative control vehicle.

-   Results: Zcyto7 had no effect on the growth of fibroblasts.

EXAMPLE 11 Effect of Zcyto7 on the Growth of BaF3 Cells

BaF3 cells, a murine pre-B cell line dependent on IL-3 to proliferate,were washed several times with base medium and then plated in a 96-wellplate each well contained approximately 5500 cells/well. The cells weretreated with either 1 μg/ml of Zcyto7 or 1-2 pg/ml of IL-3 or with acombination of both Zcyto7 and IL-3. Also in a separate experiment0.1-10 ng/ml of TGFβ was added to the wells instead of Zcyto7. Afterincubation of the assay plate at 37° C. and 5% CO2 for 3-6 days, 20 μlof ALAMAR blue was added to each well and the plate is incubated at 37°C. for 15-24 hours. The plate was then read with a fluorometer withexcitation wavelength of 544 m and emission wavelength of 590 m. Theassay was also scored by eye for stimulation or inhibition of cellproliferation prior to the addition of the ALAMAR blue. The base mediumcontained RPMI 1640+10% HIA-FBS+L-glutamine+Na pyruvate.

-   Results: Zcyto7 and TGFβ significantly inhibited the IL-3 driven    proliferation of BaF3 cells. However, when neutralizing antibodies    to TGFβ were added in along with the Zcyto7, the Zcyto7 inhibition    of the proliferation of the BaF3 cells was eliminated.

EXAMPLE 12 Effect of Zcyto7 on the Growth of TF-1 Cells

TF-1 cells, a human leukemia cell line which is GM-CSF or IL-1βdependent, were washed several times with base medium and then plated ina 96-well plate each well containing approximately 7000 cells/well. Thecells were co-cultured with 1 μg/ml of Zcyto7 and 100-200 pg/ml ofIL-1β. Also in a separate experiment TGFβ was added to the wells insteadof Zcyto7. After incubation of the assay plate at 37° C. and 5% CO2 for3-6 days, 20 μl of ALAMAR blue was added to each well and the plate isincubated at 37° C. for 15-24 hours. The plate is then read with afluorometer with excitation wavelength of 544 nm and emission wavelengthof 590 nm. The assay was also scored by eye for stimulation orinhibition of cell proliferation prior to the addition of the ALAMARblue. The base medium contained RPMI 1640+10% HIA-FBS+L-glutamine+Napyruvate.

-   Results: The IL-1β stimulation of TF-1 cells is inhibited by both    Zcyto7 and TGF-β. The concentration of Zcyto7 at which inhibition of    proliferation occurred was greater than 200 ng/ml; and the    concentration of TGF-β at which inhibition of proliferation occurred    was about 50 pg/ml.

1. An isolated polynucleotide which encodes a mammalian Zcyto7polypeptide wherein said polynucleotide encodes a polypeptide comprisedof an amino acid sequence selected from the group consisting of SEQ IDNO: 2,SEQ ID NO: 12, SEQ ID NOs:14-25 and SEQ ID NOs: 39 and 40 or apolypeptide that is at least 90% identical to said polypeptides.
 2. Thepolynucleotide of claim 1 wherein the polypeptide is fused to a carrierpolypeptide or other carrier molecule.
 3. An expression vectorcomprising the following operably linked elements: a transcriptionpromoter; a DNA segment which encodes a polypeptide comprised of anamino acid sequence selected from the group consisting of SEQ ID NO:2,SEQ ID NO: 12, SEQ ID NOs:14-25 and SEQ ID NOs: 39 and 40 or apolypeptide that is at least 90% identical to said polypeptides. ; and atranscription terminator.
 4. An expression vector comprising thefollowing operably linked elements: (a) a transcription promoter; (b) aDNA segment encoding a chimeric polypeptide, wherein said chimericpolypeptide is comprised of a first portion and a second portion joinedby a peptide bond, said first portion being comprised of a polypeptidecomprised of an amino acid sequence selected from the group consistingof SEQ ID NO: 2,SEQ ID NO: 12, SEQ ID NOs:14-25 and SEQ ID NOs: 39 and40 or a polypeptide that is at least 90% identical to said polypeptidesand said second portion being a second polypeptide or protein. (c) atranscription terminator.
 5. An isolated polypeptide comprised of anamino acid sequence selected from the group consisting of SEQ ID NO:2,SEQ ID NO: 12, SEQ ID NOs: 14-25 and SEQ ID NOs: 39 and 40 or apolypeptide that is at least 90% identical to said polypeptides.
 6. Anantibody, antibody fragment or single-chain antibody that specificallybinds to a mammalian polypeptide, said polypeptide being comprised of anamino acid sequence selected from the group consisting of SEQ ID NO:2,SEQ ID NO: 12, SEQ ID NOs: 14-25 and SEQ ID NOs: 39 and 40 or apolypeptide that is at least 90% identical to said polypeptides.
 7. Anantibody of claim 6 wherein said antibody is either monoclonal orpolyclonal.
 8. The antibody, antibody fragment or single-chain antibodyof claim 6 wherein said antibody, antibody fragment or single-chainantibody is humanized.
 9. A method for producing an antibody which bindsto a polypeptide comprised of an amino acid sequence selected from thegroup consisting of SEQ ID NO: 2,SEQ ID NO: 12, SEQ ID NOs:14-25 and SEQID NOs: 39 and 40 or a polypeptide that is at least 90% identical tosaid polypeptides comprising inoculating an animal with said polypeptideor with a nucleic acid which encodes said peptide or polypeptide,wherein said animal produces antibodies to said or polypeptide; andisolating said antibody.
 10. The method of claim 9 wherein said antibodyis either a polyclonal or monoclonal antibody.
 11. An anti-idiotypicantibody, anti-idiotypic antibody fragment or anti-idiotypicsingle-chain antibody which binds to an antibody, an antibody fragmentor single-chain antibody that specifically binds to a polypeptidecomprised of an amino acid sequence selected from the group consistingof SEQ ID NO: 2,SEQ ID NO: 12, SEQ ID NOs:14-25 and SEQ ID NOs: 39 and40 or a polypeptide that is at least 90% identical to said polypeptides.